Molecular profiling of heterogeneous circulating tumor cells (CTCs) at the single-cell level is critical for identifying different CTC subpopulations and understanding their value in predicting metastatic cancer progression and responses to treatment. Unfortunately, the isolation and comprehensive characterization of hundreds of individual CTCs present formidable analytical and technical challenges. To this end, we developed a high-throughput (HT) technology to obtain the genome-wide expression profiles of hundreds to thousands of single epithelial cancer cells in a background of contaminating leukocytes. The genetic profiling technology uses unique cell-specific molecular barcodes to label mRNAs in individual viable cancer cells to obviate the need for individual cell separation. Digital expression data are generated by HT sequencing of barcoded amplified cDNAs and easily cluster to each cell in silico using cell-specific barcodes. The goal of the Phase I contract proposal is to develop and validate a set of reagents and supporting protocols for single-cell expression profiling of the 500 most informative subtyping and metastatic signature genes in hundreds to thousands of CTCs. In the Phase II contract, the CTC expression profile analysis will be applied to subtype and molecularly profile CTCs in a large panel of patient blood samples with metastatic breast carcinomas. The ultimate goal of the project is to develop a prototype for a cost-effective prognostic diagnostic kit using single-cell multiplex amplification of the most informative biomarkers that can enable clinicians to predict the risk of metastatic relapse and to monitor the efficacy of therapies for a wide range of epithelial cancers